Notebook : August 28

summary

• We got purple clonies on ligation promoter fnr(repressor) plus RBS_AmilCP+Term_PSB1C3

so we do a purify of 4 clonies in two plate.incubated at 37°C in aerobic and anaerobic condition over night to verify the different.

• Prepare LB plates with Chlorenphenicol and Xgal .Pufify single conle of promoter fnr(activator)nirB plus RBS_LacZ+Term_PSB1C3 and promoter fnr(repressor) plus RBS_LacZ+Term_PSB1C3 to see if color will change.

• Gel extration of Digestion Dnp1 purification of the the PSB1C3 clean for Gibson and do Gibson assembly again .

lab work

A - Aerobic/Anaerobic regulation system / B - PCB sensing system

• 1 - Gel extraction of the PSB1C3 Clean for Gibson.

Protocol : Gel extraction

• 2- Migration of 3µl PSB1C3 Clean for Gibson (product gel extraction) '

• 3- product quantification.

We used the nano drop to measure the DNA in 260nm and we found its concentration

• 4 - Gibson assembly.

• RBS_BphR2_part1, BphR2_part2 and plasmid PSB1C3
• FNR_part1, FNR part1 and plasmid PSB1C3
• RBS_FNR part1, FNR_part2 and plasmid PSB1C3

Sample Volume:

These 3 mixture is incubated at 50°C for up to one hour. Transformation and spread in 6 LB plates with Chlorenphenicol . Incubate at 37°C over night.

A - Aerobic/Anaerobic regulation system

• 1 - Purify single clone of promoter fnr(activator)nirB + RBS_LacZ+Term_PSB1C3 on plates wiht Chlorenphenicol and Xgal.incubated at 37°C in anaerobic condition over night.

Protocol : Purify single clone

• 2 - Purify single clone of promoter promoter fnr(repressor)+ RBS_LacZ+Term_PSB1C3 on plates wiht Chlorenphenicol and Xgal.incubated at 37°C in aerobic condition over night

• 3 - Purify single clone of promoter fnr(activator)nirB + RBS_AmilCP+Term_PSB1C3 on plates wiht Chlorenphenicol.incubated at 37°C in anaerobic condition over night

• 4 - Purify 4 single purple colony of promoter fnr(repressor)+ RBS_AmilCP+Term_PSB1C3 on two plates wiht Chlorenphenicol .incubated at 37°C in aerobic and anaerobic condition over night

• 5- Purify single clone of B5 promoter fnr(activator)nirB + RBS_AmilCP+Term_PSB1C3 on plates wiht Chlorenphenicol.incubated at 37°C in anaerobic condition over night

• 6- Purify single clone of D1, D5, D6 promoter fnr(repressor)+ RBS_AmilCP+Term_PSB1C3 on two plates wiht Chlorenphenicol .incubated at 37°C in aerobic and anaerobic condition over night

• 7- Purify single clone of C1, C2, C5 ,C6promoter promoter fnr(repressor)+ RBS_LacZ+Term_PSB1C3 on plates wiht Chlorenphenicol and Xgal.incubated at 37°C in aerobic condition over night

Notebook : August 28

Lab work

A - Aerobic/Anaerobic regulation system

Objective : characterize Bba_K1155000 and Bba_K1155004

1 - Purification of colony transformed with ligation : NirB with RBS-LacZ-Term in PSB1C3, NirB with RBS-Amil CP-Term in PSB1C3, Pfnr with RBS-LacZ-Term in PSB1C3, Pfnr with RBS-Amil CP-Term in PSB1C3 by streaking in aerobic or anaerobic conditions

XiaoJing

A - Aerobic/Anaerobic regulation system / B - PCB sensor system

Objective : obtaining FRN and BphR2 proteins

1 - Gel purification of PSB1C3 digested by DnpI

XiaoJing

Protocol : Gel purification

Nanodrop :

• PSB1C3 : 37.2ng/µL

2 - Electrophoresis to check the gel purification of PSB1C3 digested by DnpI

XiaoJing

expected sizes :

• PSB1C3 : 2070 bp

3 - Gibson assembly

XiaoJing

Used quantities :

• RBS-BphR2 :
  • PSB1C3 : 3µL
  • BphR2 Part I : 1µL
  • BphR2 Part II : 1µL
  • Gibson mix : 15µL ???????????????

• FNR :
  • PSB1C3 : 3µL
  • FNR Part I : 1µL
  • FNR Part II : 1µL
  • Gisbon mix : 15µL ???????????????

• RBS-FNR :
  • PSB1C3 : 3µL
  • RBS-FNR Part I : 1µL
  • FNR Part II : 1µL
  • Gibson mix : 15µL ????????????

We let these mix at 50°C during 1h.